Flow cytometry is a well known means of measuring certain physical and chemical characteristics of cells or particles by sensing certain optical properties of the cells or particles as they travel in suspension, one by one past a sensing point. Flow cytometry is widely used in the biological and medical fields.
In a typical flow cytometer, the single file flow of particles is achieved using hydrodynamic focusing of the suspended particles within a sheath fluid. The particles are then individually interrogated by a light beam. In most modern cytometers, the light source is a laser which emits coherent light at a specified wavelength. Each particle interacts with the light beam, and typically the scattered and any emitted fluorescent light produced by each particle is collected. By analyzing the scattered light, physical characteristics such as cell size, shape and internal complexity can be determined. Collecting any emitted fluorescent light also allows any cell component or function that can be detected by a fluorescent compound to be examined.
Traditional flow cytometers only detect elastically scattered light (also known as “Rayleigh scattered light”) which does not contain any information about the atomic or molecular structure of the particle. Although such flow cytometers can identify the size and shape, they cannot differentiate between similarly sized, but chemically different molecules or cells, unless the molecules or cells have been tagged with fluorescent markers.
In principle, detecting the in-elastically scattered light (also known as “Raman scattered light”) would enable the chemical identification of cells or molecules, as each cell or molecule has a unique Raman spectrum based on its chemical structure. The cross-section for Raman scattering is, however, about 15 orders of magnitude lower than the cross-section for Rayleigh scattering. This means that obtaining the Raman spectrum of an individual particle in a flow cytometer is well beyond the realms of practicality because of the lack of a suitably intense light source and the lack of any suitably sensitive detectors. The Raman cross-section, however, can be dramatically increased by a technique known as surface-enhanced Raman scattering, in which the molecule or particle is placed in contact with a suitably roughened noble metal surface, or in contact with a noble metal colloidal aggregate. Under the right conditions, the cross-section of surface enhance Raman scattering approaches the cross-section for fluorescence emission.
In order for flow cytometers to be able to identify individual particle based primarily on the chemical or molecular structure of the particle, what is needed is an apparatus and method that allows particles or cells in a cytometer to be in a condition that produces a sufficiently large surface-enhanced Raman scattering cross-section so that Raman spectra can be obtained from each particle or cell as it passes the cytometer sensing point. Such a system will allow the individual identification of all cells or particles, no matter how similar in size or shape they are to each other, without any need for fluorescent tagging.